Epigenetic modifications have been recognized as important factors in modification of gene expression levels, specification of cellular state and developmental potential, and etiology of cancer. In particular, post- translational modifications to histone proteins have been shown to be a key carrier of epigenetic information. Traditional techniques of studying these epigenetic modifications, such as chromatin immunoprecipitation (ChIP), have generally focused on examination of one mark at a time. However, it is likely that multiple epigenetic signals act together to form a complex code of transcriptional and cellular regulation. Mass spectrometry is uniquely suited to studying combinations of histone modifications by directly observing them together on protein molecules or in protein complexes. In this proposal, we will adopt some strategies used in ChIP to enable multiplex recognition and quantification of epigenetic modifications to histones. As part of these efforts, we will also assess the specificity of commonly used histone modification antibody reagents for ChIP by directly probing the modifications of the histones recovered. We will extend these techniques to the study of specific loci by developing a novel hybridization-based method of selective enrichment of targeted genomic regions. Throughout these studies, the use of cross-linking (as in ChIP) will maintain the native chromatin context of the cell so that co-occurrence of histone modifications can be discovered. We expect these technologies to be applicable to detailed studies of histone modifications present at developmentally important loci, especially those required for pluripotency. PUBLIC HEALTH RELEVANCE: The study of the transmission and regulation of epigenetic information is both the natural extension of and equally important to the Human Genome Project. Furthering our understanding in this area will help us learn about the developmental potential of stem cells and enable advances in regenerative medicine.